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US20030100492A1 - Proteoglycans and pharmaceutical compositions comprising them - Google Patents

Proteoglycans and pharmaceutical compositions comprising them Download PDF

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US20030100492A1
US20030100492A1 US10/149,326 US14932602A US2003100492A1 US 20030100492 A1 US20030100492 A1 US 20030100492A1 US 14932602 A US14932602 A US 14932602A US 2003100492 A1 US2003100492 A1 US 2003100492A1
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molecule
syndecan
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Avner Yayon
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Yeda Research and Development Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

  • the present invention relates to heparan sulfate proteoglycans, particularly to syndecans, and to their several uses in promotion of tissue-specific cell proliferation, migration and differentiation.
  • FGF fibroblast growth factor
  • FGF2 basic FGF
  • FGF1 acidic FGF
  • FGFR FGF receptor
  • HS heparan sulfate
  • HSPG heparan sulfate proteoglycan
  • EDHS endothelial cells derived HSPG
  • AP alkaline phosphatase
  • FCS Dulbecco's modified Eagle medium
  • FCS fetal calf serum
  • GST glutathione S-transferase
  • MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester
  • SDS sodium-dodecyl-sulfate
  • PAGE polyacrylamide gel electrophoresis
  • PMSF phenylmethylsulfonyl fluoride
  • KLH keyhole limpet hemocyanin.
  • Fibroblast growth factors constitute a family of at least eighteen polypeptides which are mitogenic for cells of mesenchymal and neuroectodermal origin (1).
  • FGFs share 30-60% amino-acid sequence homology and a high affinity for heparin and heparan-sulfates (HS).
  • HS heparan-sulfates
  • the basic heparan sulfate proteoglycan (HSPG) structure consists of a protein core to which several linear heparan sulfate chains are covalently attached (11).
  • a few HSPGs were purified to homogeneity, including the large extra-cellular matrix HSPG perlecan (12), the membrane associated glypicans (13) and the integral membrane HSPGs, syndecan, fibroglycan (14), N-syndecan (15) and amphyglycan/ryudocan (13, 16).
  • the last four comprise a family of membrane integral HSPGs and were re-named Syndecan 1-4 (in the above same order) (17).
  • syndecans share a similar structure that includes a short highly conserved intracellular carboxy-terminal region, a single membrane-spanning domain and an extracellular domain with three to five possible attachment sites for glycosaminoglycans (17).
  • the intracellular conserved region of syndecan-4 was recently shown to interact with Protein kinase C and with phosphatidylinositol 4,5-biphosphate, both of which can direct and regulate the recruitment of syndecan-4 to the cells focal contacts (18-20).
  • perlecan the large basement membrane HSPG (12) isolated from human fetal lung fibroblasts, was found to induce high affinity binding of FGF2 to FGFR1 as well as to promote FGF dependent angiogenesis in vivo (23). More recently syndecan-2 isolated from macrophages was found to enhance receptor and biological activity of FGF2 (24).
  • binding of fibroblast growth factors (FGFs) to their high affinity receptors is potentiated by heparin or heparan sulfate (HS).
  • FGFs fibroblast growth factors
  • HSPG integral membrane heparan sulfate proteoglycans
  • syndecans When expressed in mutant cells, deficient in total HS or which specifically lack 2-O-sulfated iduronic acids, syndecans do not support receptor binding of FGF1 or 2.
  • Syndecan-4 was also found to form SDS-resistant dimers, similar to those observed for syndecans-1 and 3, the formation of which we find to be partially dependent on its HS chains.
  • the present invention thus relates to a molecule capable of promoting high affinity binding of a fibroblast growth factor (FGF) to a FGF receptor (FGFR), said molecule being selected from:
  • a recombinant chimeric fusion molecule comprising the extracellular domain of a syndecan or a fragment thereof fused to a tag suitable for proteoglycan purification, said fusion molecule being post-translationally glycosylated to carry at least one chain of a beparan sulfate having at least one highly sulfated domain;
  • the molecule according to the invention may promote high affinity binding of FGF1 and FGF2 to FGFR1, or of FGF9 to FGFR2 and to FGFR3, or of any other FGF to its respective receptor(s).
  • the extracellular domain according to (i) and (ii) above may be an extracellular domain of any of the syndecans -1, -2, -3 or -4, or a fragment thereof, wherein said extracellular domain or fragment preferably comprises the glycosylation sites of the syndecan molecule.
  • the extracellular domain comprises the amino acids 1-145 of syndecan-4, and a fragment thereof comprises at least 75 amino acids of the extracellular domain of syndecan-4.
  • the syndecan extracellular domain may be fused to any tag suitable for proteoglycan purification including, but not being limited to, glutathione S-transferase (GST) or polyHis, and preferably the Fc region of the human gamma globulin heavy chain.
  • GST glutathione S-transferase
  • polyHis polyHis
  • the post-translational glycosylation occurs when a DNA molecule according to (ii) above is expressed in suitable mammalian cells including, but not being limited to, endothelial, fibroblast, and epithelial cells, such as embryonic kidney cells, ovary cells, e.g. chinese hamster ovary cells (CHO), or aortic endothelial cells.
  • suitable mammalian cells including, but not being limited to, endothelial, fibroblast, and epithelial cells, such as embryonic kidney cells, ovary cells, e.g. chinese hamster ovary cells (CHO), or aortic endothelial cells.
  • ovary cells e.g. chinese hamster ovary cells (CHO)
  • CHO chinese hamster ovary cells
  • glycosaminoglycan chains of syndecans according to (iii) above may be prepared by protease treatment of the syndecan, for example as described in Nader et al., 1987 (27).
  • the heparan sulfate that constitutes the glycosyl chain of the syndecan has, preferably, at least one highly O-sulfated domain of at least 10 sugar units, and is preferably 2-O-sulfated.
  • Syndecan coding sequences may be obtained by cDNA cloning or by reverse transcriptase PCR cloning by standard methods well known in the art.
  • the desired extracellular domain or fragments thereof can then be excised by restriction enzyme digest or by PCR using appropriate oligonucleotide primers.
  • the so obtained sequences may then be fused to a suitable tag to form the DNA sequences of (ii) above, preferably with the Fc of the immunoglobulin heavy chain, most preferably human IgG1.
  • the ectodomain of the syndecan When expressed as a fusion protein, the ectodomain of the syndecan will usually be cleaved from the fusion partner. This expression may occur in vivo after administration of a DNA sequence of (ii) above, thus making the soluble biologically active extracellular domain of the syndecan available to exert the desired biological activity.
  • the recombinant chimeric fusion molecule comprises the extracellular domain of syndecan- 1, -2, -3, or -4 fused to the recombinant Fc region of the human gamma globulin heavy chain, carrying at least one chain of a heparan sulfate having at least one highly sulfated domain (Syn1-Fc, Syn2-Fc, Syn3-Fc, Syn4-Fc).
  • the chimeric molecule may carry 1, 2 or the 3 polysaccharide chains of Syn4.
  • the chimeric fusion molecule of (i) above, the DNA molecule of (ii) above and the syndecan derived sugar molecule of (iii) above are capable of modulating (both enhancing and inhibiting) heparin-dependent growth factor activity relevant for promoting tissue-specific cell proliferation, migration and differentiation.
  • the growth factor which activity can be modulated by said molecule includes, but is not limited to, a FGF, a vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), an epidermal growth factor (EGF) and keratinocyte growth factor (KGF).
  • the present invention thus further relates to pharmaceutical compositions comprising a molecule (i), (ii) or (iii) of the invention and a pharmaceutically acceptable carrier.
  • This composition can be used for induction of angiogenesis, bone fracture healing, enhancement of wound healing, promotion of tissue regeneration and treatment of ischemic heart diseases and of peripheral vascular diseases, for example for promoting liver regeneration, or for promoting tissue regeneration alter transplantation of myocytes into heart tissues, or after transplantation of cells into brain tissue.
  • the molecules of the invention can further be used in combination with one or more growth factors such as a FGF, e.g. FGF2, a VEGF, an EGF, HGF and/or KGF.
  • the growth factor may be administered before, together with, or after the molecule of the invention.
  • a molecule of the invention may be administered together with: (a) FGF2 for treatment of heart failure by transplantation of myocytes, or for promotion of tissue regeneration after transplantation of dopaminergic/neuronal cells for example in Parkinson disease; (b) FGF2 and/or VEGF for induction of angiogenesis or for treatment of ischemic heart disease or peripheral vascular disease; (c) HGF for promoting liver regeneration; (d) KGF for enhancement of wound healing.
  • FIG. 1 shows that binding of FGF2 and FGF1 is modulated by purified endothelial derived syndecan-4.
  • Soluble extracellular domain of FR1-AP fusion protein was immunoprecipitated with anti-alkaline phosphatase antibodies and incubated with 125 I-FGF1 (right panel) or 125 I-FGF2 (left panel), in the absence or presence of 1 ⁇ g/ml heparin, endothelial derived syndecan-4 (EDHS), or the isolated syndecan-4 HS chains. Binding was performed as described under ‘Experimental Procedures’. Bound complexes were extensively washed with low affinity buffer to remove FGFs bound to the HS. The associated radiolabeled FGFs were determined by a gamma-counter. These results represent one out of three independent experiments, carried out in duplicates. Standard error bars are indicated.
  • FIGS. 2 A- 2 B show overexpression of syndecan-4 in CHO-KI cells.
  • FIG. 2A Confluent cultures of wild type CHO-KI cells transfected with syndecan-4 cDNA were incubated with specific monoclonal antibodies directed to the extracellular domain of syndecan-4, and detected by radiolabeled anti-mouse antibodies (filled bars). Cells were lysed and counted in a gamma-counter. CHO-KI cells of the identified syndecan-4 positive clones were metabolically labeled with 35 S-sulfuric acid for 24 hours and the amount of heparan sulfate associated radioactivity was measured by liquid scintillation (dashed bars) as described under ‘Experimental Procedures’.
  • FIG. 2B Positive clones of wild type CHO-KI and GAG deficient mutant CHO-745 were extracted as described under ‘Experimental Procedures’ and treated with heparinase-I and -II mixture. Syndecan-4 protein bands were examined by running equal amounts of cell extracts on SDS-PAGE and transferring to a nitrocellulose membrane. Detection was done with P710 anti-syndecan-4 polyclonal antibodies.
  • FIGS. 3 A- 3 B show binding of FGF2 to FGFR1 on immobilized syndecan-4.
  • Cells of the indicated clones were extracted as described under ‘Experimental Procedures’. Equal amounts of cell extracts were immunoprecipitated with anti-P710 antibodies and incubated with FGF2 (50 ng).
  • FIG. 3A Proteins were separated on reducing SDS-PAGE containing ⁇ -mercaptoethanol, and transferred to a nitrocellulose membrane. FGF2 was detected with the FB-8 monoclonal antibody. Minor amounts of FGF2 were non-specifically bound to the beads (right lane). D-dimers; M-monomers.
  • FIGS. 4 A- 4 C show expression and metabolic labeling of a soluble secreted Syn4-Fc fusion protein.
  • FIG. 4A The extracellular domain of syndecan 4 cDNA (black) was subcloned into the CDM7 vector in frame with the Fc portion of human gamma globulin (doted). The BamHI and HindIII sites used for cloning are indicated.
  • FIG. 4B CDM7-Syn4-Fc plasmid was co-transfected with the pcDNA3 neomycin resistant vector into 293T cells, and positive clones were selected by dot-blot analysis.
  • FIG. 4C Positive 293T clones expressing the Syn4-Fc fusion protein were metabolically labeled with 35 S-sulfuric acid and 3 H-leucine for 24 hours The conditioned medium was collected and concentrated on Protein-A Sepharose. Equal amounts of radiolabeled syndecan-4 from each of the different clones (Table 1) was separated on a 3-15% gradient SDS-PAGE without or with pre-treatment with heparinase-I and -III (Hepa's). The gel was dried and exposed to X-ray Kodak film for 3 days.
  • FIGS. 5 A- 5 C show that syn4-Fc promotes the binding and mitogenic response to FGF2 and FGF1.
  • FIG. 5A High affinity binding of FGF2 and FGF1 to FGFR1.
  • Conditioned media 100 ⁇ l
  • 293T cells expressing Syn4-Fc or Erb4-Fc was immobilized on Protein A Sepharose and incubated in the absence or presence of 75 ng of either FGF1 or FGF2.
  • the coupled beads were washed with HNTG, further incubated with FR1-AP for 2 hours and extensively washed. The bound receptor level was determined by the associated AP activity.
  • FIG. 5A High affinity binding of FGF2 and FGF1 to FGFR1.
  • Conditioned media 100 ⁇ l
  • the coupled beads were washed with HNTG, further incubated with FR1-AP for 2 hours and extensively washed.
  • the bound receptor level was determined by the associated AP activity.
  • FIG. 5B The ability of conditioned media (100 ⁇ l) from 293T cells expressing Syn4-Fc either untreated or treated with heparinase-I and -III (Hepa's), to promote binding of FGF2 to FR1-AP, is indicated.
  • FIG. 5C Syn4-Fc promotes FGF1 dependent mitogenic response of FGFR1 expressing cells. Thymidine incorporation into heparan sulfate deficient (745) CHO cells overexpressing FGFR1.
  • FIG. 6 shows syndecan- 1, -2 and -4 Fc specific induction of FGF-FGFR binding.
  • Conditioned media of growth plate derived chicken chondrocytes cells (LSV) expressing the chimeric Syndecans 1, 2, 3 or 4 fused to the human IgG-Fc fragment were incubated with protein A-agarose beads. The beads were then washed with 2M NaCl and incubated with FGF1, FGF2 or FGF9, following by incubation with soluble FGF receptors (FGFR) 1, 2 and 3, fused to human placental alkaline phosphatase. Significant differences in the binding specificity of the different FGF-FGFR complexes exist.
  • LSV growth plate derived chicken chondrocytes cells
  • Syn-4-Fc promotes the interaction of FGF2 with FGFR1 and FGFR2 but not with FGFR3.
  • Syn-2-Fc promotes the interaction of all tested ligands with FGFR3 but not all other tested interactions.
  • Syn-1-Fc a high affinity interaction of FGF2 with FGFR3, which was not observed with the other syndecans or with cells expressing FGFR3.
  • FIGS. 7 A- 7 B show the effects of 2-O-sulfation on syndecan-4 activity.
  • FIG. 7A Positive 293T or Pgs-F17 clones expressing the Syn4-Fc fusion protein were metabolically labeled with 35S-sulfuric acid for 24 hours. The conditioned medium was collected and concentrated on Protein A-Sepharose. Equal amounts of radiolabeled syndecan-4 from each of the different clones (Table 1) were separated on a 3-15% gradient SDS-PAGE. The gel was dried and exposed to X-ray Kodak film for 3 days.
  • FIG. 7A Positive 293T or Pgs-F17 clones expressing the Syn4-Fc fusion protein were metabolically labeled with 35S-sulfuric acid for 24 hours. The conditioned medium was collected and concentrated on Protein A-Sepharose. Equal amounts of radiolabeled syndecan-4 from each of the different clones (Table 1) were separated on a 3-15% gradient SDS-PAGE. The
  • Conditioned media (100 ⁇ l) from the above clones was adsorbed to Protein-A Sepharose incubated without or with 75 ng of FGF1 or FGF2, as indicated.
  • the coupled beads were washed with HNTG, further incubated with FR1-AP for 2 hours and extensively washed.
  • the bound receptor level was determined by the AP activity. Each data point is the mean of duplicate determinations.
  • FIG. 8A depicts the nucleotide and amino acid sequences of syndecan-4.
  • the nucleotide sequence of mouse EDHS (syndecan-4 homologue) and its deduced amino acid sequence in one letter code are shown.
  • the single putative transmembrane domain is underlined.
  • the potential glucosaminoglycans attachment sites are indicated by diamonds ( ⁇ ).
  • the doted underline indicates the sequence of the peptide P710 used as antigen for antibody preparation.
  • FIG. 8B Amino acids sequences of mouse syndecan-1 (49), rat syndecan-2 (50), mouse syndecan-3 (14) and mouse syndecan-4 were compared using the GCG pileup program.
  • Black background indicates at least three identical amino acids, and gray background indicates at least three similar amino acids.
  • FIG. 8C Amino acids sequences of syndecan-14 from mouse (EDHS, FIG. 8A), rat (ryudocan), human (amphiglycan) and chicken were compared using the GCG pileup program. Black background indicates at least three identical amino acids, and gray background indicates at least three similar amino acids.
  • syndecan-4 expressed either as an integral transmembrane proteoglycan or in a soluble secreted form efficiently enhanced high affinity binding of both FGF1 and FGF2. This effect of syndecan-4 was not restricted to FGFR1 but was shown to occur also with FGFR2. These results indicate that syndecan-4 plays an important role in regulating FGF-FGFR binding and signaling in vivo.
  • Perlecan the large basement membrane HSPG was previously found to induce high affinity binding and biological activity of FGF2 (23). More recently glypican isolated from rat embryonal myoblasts (39) and syndecan-1 expressed in Raji lymphoma cells (40) were shown to mediate FGF binding and activity. This may imply some functional redundancy with regard to activation of FGFs by multiple, nevertheless discrete, types of HSPGs from the cell surface and the extracellular matrix. Alternatively, there may be a specific effect for each proteoglycan that is at least partially determined by the localization of the proteoglycan in either the extracellular matrix or at the cell surface.
  • proteoglycans may act in synergism to enhance specific activation by FGFs.
  • FGF2 mitogenic activity 41). This co-amplification may serve as an example of a coordinated action of cell surface and extracellular matrix activating proteoglycans that act in concert to enhance FGF signaling.
  • an activating HSPG on the cell surface may be of special importance for the autocrine activity of FGF.
  • Such an autocrine activity has been proposed to regulate endothelial cell proliferation and to drive autocrine growth in several melanoma cell lines that produce FGF2 and are dependent on endogenous FGF2, in contrast to normal melanocytes (42). Transformation of NIH-3T3 cells by signal peptide containing FGFs has also been suggested to result from an internal autocrine signaling loop (43, 44).
  • a basic characteristic of this autocrine activity is that all components of the signaling complex including the appropriate HSPG should be expressed within the same cell. Syndecan-4 expression is highly abundant in vivo and is found on a variety of cell lines including endothelial, neural, fibroblastic and epithelial cells (45) where it can serve as an integral part of such an FGF autocrine complex.
  • syndecan-4 is solely dependent on its HS chains, therefore, eliminating these chains either by heparinase treatment or by expressing the core protein in the Pgs-A745 CHO mutant cell line, completely abolished its effect.
  • the nature and defined structure of the glycosaminoglycan chains could, in principle, be determined by the nature of the core protein carrying these chains or alternatively by the type and differentiation stage of the cells expressing these core proteins.
  • syndecan-4 (or its ectodomain) in different cell types including endothelial, fibroblast or epithelial cells results in a recombinant proteoglycan that can bind FGF2 and share a similar capability to promote a high affinity interaction with FGFR1.
  • heparin required to promote high affinity binding of FGF2 are specific and restricted to highly O-sulfated oligosaccharides of at least 10 sugar units in length (21, 34, 35).
  • Heparin and HS fragments with high affinity for FGF2 and FGF1 were isolated and found to be polymers rich in 2-O-sulpho- ⁇ -L-iduronic acid (46, 47). These specific domains of high charge density, while widely distributed in heparin, are rare in HS, where they may be involved in FGF binding and activation.
  • the HS structure determined for syndecan-4 associated HS chains, isolated from endothelial cells, is composed of four highly sulfated, heparin like domains (27).
  • Each of these contains two regions rich in iduronic acid tri- and disulfated disaccharides and tetra- and pentasulfated tetrasaccharids typical of heparin.
  • expression of syndecan-4 in cells incapable of proper 2-O-sulfation results in a proteoglycan that fails to promote FGF2-receptor interaction, supporting the notion that 2-O-sulfated iduronic acid rich domains in HS are crucial for its FGF promoting activity.
  • syndecan-4 Overexpression of syndecan-4 in wild type CHO cells results in self-association of the core protein and the formation of SDS resistant dimers. A similar phenomenon was reported for syndecan-3, where self-association was suggested to be mediated by a unique structural motif in the protein transmembrane domain (33). This domain is highly conserved among, the different syndecans and may, therefore, share a similar function in syndecan-4 as well. No dimers or higher order oligomers of soluble syndecan-4, lacking the transmembrane domain were detected, suggesting that indeed the sequence responsible for self-association reside within the transmembrane or intracellular domain of syndecan-4.
  • syndecan-4 Another, most likely related finding regarding syndecan-4, is the recent discovery that it is selectively enriched in focal adhesion contacts (48).
  • a role for HSPGs in adhesion was previously suggested, based on the finding that adhesion defective cells have cell surface HSPGs of altered properties (49).
  • the recruitment of syndecan-4 into focal contacts appears to be coordinately regulated by protein kinase-C (18) and phosphatidylinositol 4,5-biphosphate (19, 20). This recruitment involves direct association and phosphorylation of the C-terminus of syndecan-4 (50) and may serve to stabilize this region.
  • FGFR1 like several other receptor tyrosine kinases, is found to be enriched in focal contacts (51).
  • syndecan-4 This co-localization of both FGFRs and accessory HSPGs such as syndecan-4 may serve as means for the local amplification of FGF signals.
  • a role for FGF signaling in the stabilization of the focal contact structure can be suggested.
  • syndecan-4 is a primary response gene induced by FGF2 (52).
  • FGF dependent modulation of focal contacts can drastically affect the adhesion and shape properties of the cell, which in turn may contribute to the well known effects of FGF on cell motility, migration and proliferation in a variety of biological processes such as wound healing and angiogenesis.
  • Heparin was obtained from Hepar Industries (Franklin, Ohio). Recombinant human FGF2 and FGF1 were kindly provided by American Cyanamid Company (Pearl River, N.Y.). Growth factors were iodinated by the chloramine T method as described previously (25). The specific activity was 1.2-1.7 ⁇ 10 5 cpm/ng and the labeled preparation was stored for up to 3 weeks at ⁇ 70° C. Heparinase III and I were purchased from Sigma (St. Louis, Mo.).
  • F12 and Dulbecco's modified Eagle's medium (DMEM), calf serum, fetal calf serum (FCS), penicillin, and streptomycin were obtained from Biological Industries (Beit-Haemek, Israel).
  • G418 was purchased from GibcoBRL (Getthersb, Md.). Tissue culture dishes were purchased from Falcon Labware Division, Becton Dickinson (Oxnard, Calif.). Na 125 I and H 2 35 SO 4 were purchased from Amersham (Buckinghamshire, England).
  • Triton X-100, nonidet P-40, para-nitro-phenyl phosphate, and all other chemicals were of reagent grade, and purchased from Sigma (St. Louis, Mo.).
  • Anti-FGF2 monoclonal antibody, FB-8 was obtained from Sigma (Israel).
  • Cell lines Wild type chinese hamster ovary cells (CHO-KI), glycosaminoglycan deficient (Pgs-A745) or 2-O-sulfated heparan deficient mutants (Pgs-F17) were cultured in F12 medium supplemented with 10% FCS.
  • NIH-3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% bovine calf serum.
  • DMEM Dulbecco's modified Eagle's medium
  • Human embryonal kidney cells (293T) were cultured in DMEM supplemented with 10% FCS.
  • Syndecan-4 was isolated from the conditioned medium of rabbit aortic endothelial cells by Sepharose CL-6B gel filtration followed by ion exchange chromatography on DEAE-cellulose as previously described (26, 27). The identity of the purified proteoglycan was confirmed by N-terminal sequencing (26).
  • Syndecan-4 cDNA Two oligonucleotide primers derived from Syndecan-4 sequence were synthesized; the forward primer, EDF. 5′-CCCAAGCTTTGTGCTGTTGGAACCATGG, and reverse primer EDB: 5′-GCGGATCCGCCTCATGCGTAGAACTCG) having Hind III and BamH I restriction sites at their 5′ ends, (underlined), respectively.
  • the primers were used for PCR amplification (35 cycles of 1 min denaturation at 94° C., annealing for 2 min at 48° C., elongation for 1 minute at 72° C.) with several cDNA libraries (from human placenta, human carcinoma, mouse brain, and mouse liver) used as templates.
  • the amplified products were resolved on a 1% agarose gel stained with ethidium bromide.
  • a PCR fragment of the anticipated size (600 bp) amplified from mouse liver cDNA library was digested with Hind III and BamH I and subcloned into pBluescript KS+ (Stratagene, Calif.). The identity of the amplified fragment was determined by sequencing.
  • mice syndecan-4 cDNA was excised from pBluescript KS+ by Xho I and Xba I and subcloned into the same sites of the pLSV mammalian expression vector (28).
  • Syndecan-4 in the pLSV expression vector was co-transfected into CHO-KI and Pgs-A745 cells, with a selectable neomycin resistance gene, by the calcium phosphate method.
  • Clones were selected in G418 (0.5 mg/ml) and screened for syndecan-4 expression by direct binding of antibodies directed against the extracellular domain of syndecan-4, or by metabolic labeling of cells with 35 S-sulfuric acid (150 ⁇ Ci for 24-48 hours).
  • the EDF forward primer and the reverse primer EDMB 5′-CGGGATCCTCAGTTCTCTCAAAGATG that contains a BamH I site (underlined).
  • the purified PCR product was cut with Hind III/BamH I and subcloned in frame to a Fc portion (including the hinge region, CH2 and CH3 domains) of human IgG1, in the CDM7 vector, to create the fusion protein Syn4-Fc.
  • the Syn4-Fc plasmid was co-transfected into 293T cells with the neomycin resistance gene, by electroporation using Gene Pulser (Bio-Rad, Calif.) set at 960 ⁇ F and 250 V. Individual clones were selected with G418 (0.6 mg/ml) and screened for Fc secretion by dot-blot of conditioned media (100 ⁇ l) with horse-radish-peroxidase (HRP) coupled anti-human Fc antibody (Sigma, Israel). The chimeric molecules with syndecan- 1, -2 and -3 were obtained in the same way.
  • Gene Pulser Bio-Rad, Calif.
  • the animals were bled and the titer and specificity of the antiserum were determined by immunoprecipitation of HSPGs from labeled lysates of human fetal lung fibroblasts and by competition for binding to syndecan-4 by the specific peptide.
  • An IgG fraction was isolated on a Protein A column according to the manufacturer's instructions (Repligen, Mass.).
  • Syndecan-4 was extracted from overexpressing cells in lysis buffer (150 mM NaCl, 20 mM Tris pH 8.0, 1 mM MgCl 2 , 0.1 mM ZnCl 2 , 0.5% NP-40, 1 ⁇ g/ml aprotinin, 1 ⁇ g/ml leupeptin, 2 mM PMSF) and cell lysates were clarified by centrifugation. Total cell extracts (100 ⁇ g protein) were immunoprecipitated with polyclonal anti-syndecan-4 antibody (P710).
  • Syn4-Fc fusion protein was immobilized directly on Protein A-Sepharose.
  • FGF 50 ng
  • FGF 50 ng
  • the beads were washed extensively with HNTG (150 mM NaCl, 10% glycerol, 0.1% Triton-X-100 and 50 mM Hepes pH 7.4) and incubated for 2 hours with conditioned media containing the soluble FGFR1 or FGFR2-alkaline phosphatase (FR1-AP and FR2-AP, respectively) fusion proteins (29-31) followed by a 0.5 M wash to eliminate non specific binding of the receptor to HS.
  • HNTG 150 mM NaCl, 10% glycerol, 0.1% Triton-X-100 and 50 mM Hepes pH 7.4
  • Alkaline phosphatase activity was monitored spectrophotometrically at 405 nm using para-nitro-phenyl phosphate as a substrate, as described (29). The extent of soluble FR-AP binding was determined by measuring alkaline phosphatase activity associated with the beads after extensive washing with HNTG.
  • Binding of FGFs to immobilized FR1-AP—FR1 -AP and FR2-AP fusion proteins were immunoprecipitated with anti AP antibodies and incubated for 4 hours with 125 I-FGF1 and 125 I-FGF2, in the presence or absence of 1 ⁇ g/ml heparin, syndecan-4 or HS-chains, in binding buffer (1% BSA and 25 mM Hepes in DMEM). The binding medium was then discarded and the cells were washed twice with binding buffer and once with 0.5M NaCl in 25mM Hepes pH 7.5. High affinity bound FGFs were eluted with a buffer of 1.6 M NaCl in 20 mM Sodium Acetate pH 4.5 and counted in a ⁇ -counter.
  • 35 S-sulfate and 3H-leucine labeling of cells Post-confluent cultures in 24-well plate were incubated for 24-36 hours in the appropriate medium supplemented with 10% fetal bovine serum, containing 20 ⁇ Ci/ml of H 2 35 SO 4 or 10 ⁇ Ci/ml of 3 H-leucine. The cells were washed twice with PBS, and scraped in a small volume of lysis buffer. The cell lysates were clarified by centrifugation and the amount of radioactive material in the pellet was measured by liquid scintillation. Alternatively, if soluble Syn4-Fc was labeled, the conditioned medium was collected and the protein was separated on Protein A-Sepharose.
  • Ectopically expressed mouse syn(lecan-4 is post-translationally modified and expressed as a cell surface HSPG—In order to study the role of syndecan-4 and its HS chains in modulating FGF-receptor interactions, mouse syndecan-4 was overexpressed in CHO-KI and in Pgs-A745-CHO mutant cells deficient in glycosaminoglycans. Positive clones identified by direct binding of monoclonal anti-syndecan-4 antibodies were selected and further tested for expression by immunoblotting (not shown). Measuring radioactive sulfate incorporated into syndecan-4 expressing CHO-KI clones normalized for total syndecan-4 (FIG.
  • Syndecan-4 binds FGF2 and promotes its binding to FGFR1—Ectopically expressed syndecan-4 efficiently binds FGF2 in vitro as demonstrated by co-precipitation of the proteoglycan and detection by immunoblot with specific anti-FGF2 antibodies (FIG. 3A).
  • Immunoprecipitated syndecan-4 from clone KI-E10 binds approximately 3-fold more FGF2 than syndecan-4 from the CHO-KI parental cells.
  • the ratio of dimers to monomers of FGF2 is higher in the KI-E10 IP, indicating that syndecan-4 not only binds FGF2 but can also enhance its dimerization.
  • FGF2 bound to syndecan-4 was also bound with high affinity to FGFR1 (FIG. 3B). Binding of FGF2 to immobilized FGFR-1 was tripled in the presence of syndecan-4 isolated from clone E10 overexpressing the ectopic proteoglycan.
  • Soluble chimeric syndecan-4 is post-translationaly modified and can modulate FGF-receptor interactions—In order to further study the effects of syndecan-4 on ligand-receptor interactions, a chimeric protein (Syn4-Fc), in which the extracellular part of syndecan-4 was fused to the Fc portion of human IgG1, was generated. The Syn4-Fc was secreted into the conditioned medium of transfected 293T cells, and isolated using Protein A chromatography. SDS-PAGE analysis of conditioned medium from transfected cells, pretreated with heparinase, revealed three protein bands that can be detected by labeled anti-human Fc antibodies (FIG. 4A).
  • a major protein band migrated at ⁇ 60 kDa, somewhat higher than the expected molecular weight of the chimeric fusion protein. This is consistent with the abnormal migration pattern observed for the full length core protein the two additional bands at 33 and 50 kDa represent most likely the Fc portion and a partial degradation product of the fusion protein, respectively.
  • the Syn4-Fc chimeric protein is post-translationally modified by HS chains as was demonstrated by metabolic labeling with 35 S-sulfate (FIG. 4B).
  • Co-labeling with 3 H-leucine and H 2 35 SO 4 enabled us to estimate the relative amount of protein and sugar in the chimeric proteoglycan by measuring radioactivity with the appropriate energy window for each isotope ( 35 S or 3 H).
  • the results are summarized in Table 1.
  • the ratio between the two isotopes is 1.54 ⁇ 0.07 for all the samples, indicating that there is a constant ratio of sulfated sugar to protein in all the selected Syn4-Fc secreting clones.
  • Syn4-Fc The activity of Syn4-Fc appeared to be specific to the syndecan-4 part of the fusion protein, as Fc coupled to the extra-cellular part of the Erb4 receptor, used as a control, did not support FR1-AP binding.
  • no association of FGF2 and soluble FGFR1 with Syn4-Fc produced in HS deficient cells could be detected (not shown).
  • Syn4-Fc was also capable of promoting the direct binding of 125 I-FGF2 to soluble FGFR2-AP (not shown).
  • Syndecan-1, -2, -3 and -4 mediate selective binding of FGFs to FGF receptors—The ability of several FGFs to interact with FGF receptors when immobilized on Syn-1, -2, -3 and -4-Fc was compared to their capacity to form specific FGF/FGFR complexes on heparin sepharose.
  • Syn4-Fc preferentially promotes the interaction of bFGF with FGFR1, and with about 2 fold less to FGR2, as measured by alkaline phosphatase activity and cross-linking of the receptors to radio-labeled bFGF. A similar activity was found for aFGF.
  • Syn4-Fc promotes FGF1 mediated proliferation of FGFR1 expressing cells—
  • FGFR1 expressing cells To study the capacity of syndecan-4 to elicit a heparin-dependent biological response to FGF, we made use of the HS deficient pgs-A745-CHO cells, transfected with FGFR1. These cells were previously shown to efficiently bind FGF2 only in the presence of heparin (25). As shown in FIG. 5C, the cells did not respond to FGF1 in the absence of heparin as measured by DNA synthesis. However, upon the addition of either heparin or purified Syn4-Fc, a clear mitogenic response to FGF1 is observed.
  • Syn4-Fc in Pgs-F17 cells, a mutant CHO cell line deficient of 2-O-sulfotransferase (37).
  • Syn4-Fc produced by these cells had a lower molecular weight (FIG. 6A) and a dramatically reduced affinity towards FGF2.

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US20050187150A1 (en) * 2001-10-31 2005-08-25 New York University Structure-based design and synthesis of FGF inhibitors and FGF modulator compounds
US20060183712A1 (en) * 2005-02-17 2006-08-17 The Texas A&M University System Affinity purified heparin/heparan sulfate for controlling the biological activity of the FGF receptor
US20080119403A1 (en) * 2004-03-31 2008-05-22 National Institute Of Advanced Industrial Science Haparin-Binding Protein Modified with Heparan Sulfate Sugar Chains, Process for Producing the Same and Pharmaceutical Compositions Containing the Same
US20090297479A1 (en) * 2008-03-28 2009-12-03 Kiyoshi Ariizumi Dc-hil conjugates for treatment of t-cell disorders
US20120039912A1 (en) * 2009-04-15 2012-02-16 Galapagos Sasu Rspondin-3 inhibition in bone disorders
US11903997B2 (en) * 2015-03-20 2024-02-20 Orbsen Therapeutics Limited Modulators of syndecan-2 and uses thereof
US11918687B2 (en) 2016-01-15 2024-03-05 Orbsen Therapeutics Limited SDC-2 exosome compositions and methods of isolation and use

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AU2002326112A1 (en) * 2001-08-09 2003-02-24 Yissum Research Development Company Of The Hebrew University Of Jerusalem Cd44 variants carrying heparan sulfate chains and uses thereof
KR20070006714A (ko) * 2003-12-29 2007-01-11 센텔리옹 에스아에스 관상 동맥 또는 말초 허혈 치료
WO2005079817A1 (fr) * 2004-02-18 2005-09-01 The Texas A & M University System Heparine/heparane sulfate purifie a affinite destine a la regulation de l'activite biologique du recepteur fgf
US20090220588A1 (en) * 2008-02-21 2009-09-03 Massachusetts Institute Of Technology Simultaneous Delivery of Receptors and/or Co-Receptors for Growth Factor Stability and Activity
US20220380489A1 (en) * 2019-10-02 2022-12-01 Kyushu University, National University Corporation Method for producing heparin-like substance, recombinant cell, and method for producing the same
GB202110693D0 (en) * 2021-07-26 2021-09-08 Univ London Queen Mary Peptides

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US5486599A (en) * 1989-03-29 1996-01-23 The Board Of Trustees Of The Leland Stanford Junior University Construction and use of synthetic constructs encoding syndecan
US5851993A (en) * 1994-06-13 1998-12-22 Biotie Therapies Ltd. Suppression of tumor cell growth by syndecan-1 ectodomain
US6368811B1 (en) * 1997-04-25 2002-04-09 Vlaams Interuniversitair Instituut Voor Biotechnologie Syndecan interacting proteins and the use thereof

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050187150A1 (en) * 2001-10-31 2005-08-25 New York University Structure-based design and synthesis of FGF inhibitors and FGF modulator compounds
US20080119403A1 (en) * 2004-03-31 2008-05-22 National Institute Of Advanced Industrial Science Haparin-Binding Protein Modified with Heparan Sulfate Sugar Chains, Process for Producing the Same and Pharmaceutical Compositions Containing the Same
US7741078B2 (en) * 2004-03-31 2010-06-22 National Institute Of Advanced Science And Technology Heparin-binding protein modified with heparan sulfate sugar chains, process for producing the same and pharmaceutical compositions containing the same
US20060183712A1 (en) * 2005-02-17 2006-08-17 The Texas A&M University System Affinity purified heparin/heparan sulfate for controlling the biological activity of the FGF receptor
US20090297479A1 (en) * 2008-03-28 2009-12-03 Kiyoshi Ariizumi Dc-hil conjugates for treatment of t-cell disorders
US20120039912A1 (en) * 2009-04-15 2012-02-16 Galapagos Sasu Rspondin-3 inhibition in bone disorders
US11903997B2 (en) * 2015-03-20 2024-02-20 Orbsen Therapeutics Limited Modulators of syndecan-2 and uses thereof
US11918687B2 (en) 2016-01-15 2024-03-05 Orbsen Therapeutics Limited SDC-2 exosome compositions and methods of isolation and use

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